title: Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na(+)-coupled phosphate cotransporter.
abstract: A common structural feature characterises sodium-coupled inorganic phosphate cotransporters of the SLC34 family (NaPi-IIa/b/c): a pair of inverted regions in the N- and C-terminal halves of the protein. These regions are hypothesised to contain re-entrant domains that associate to allow alternating access of the substrates from either side of the membrane. To investigate if these domains interact during the NaPi-II transport cycle, we introduced novel cysteines at three functionally important sites associated with the predicted re-entrant domains of the flounder NaPi-IIb for the purpose of fluorescent labelling and cross-linking. Single and double mutants were expressed in Xenopus oocytes and their function analysed using electrophysiological and real-time fluorometric assays. The substitution at the cytosolic end of the first re-entrant domain induced a large hyperpolarizing shift in the voltage dependence of steady-state and presteady-state kinetics, whereas the two substitutions at the external face were less critical. By using Cu-phenanthroline to induce disulfide bridge formation, we observed a loss of transport activity that depended on the presence of sodium in the incubation medium. This suggested that external sodium increased the probability of NaPi-IIb occupying a conformation that favours interaction between sites in the re-entrant domains. Furthermore, voltage-dependent fluorescence data supported the hypothesis that a localised interaction between the two domains occurs that depends on the membrane potential and substrate present: we found that the fluorescence intensity reported by a labelled cysteine in one domain was dependent on the side chain substituted at a functionally critical site in the opposed domain.
authors.affiliation: Institute of Physiology and ZIHP, University of Zurich, Winterthurerstrasse, Switzerland.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ghezzi
authors.forename: Anne-Kristine
authors.initials: AK
authors.lastname: Meinild
authors.forename: Heini
authors.initials: H
authors.lastname: Murer
authors.forename: Ian C
authors.initials: IC
authors.lastname: Forster
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D005432:Flounder; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D050608:Sodium-Phosphate Cotransporter Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D050608:Sodium-Phosphate Cotransporter Proteins; D003545:Cysteine